A large body of evidence demonstrates that the Snf1-related protein kinases serve as important regulators modulating fundamental metabolic pathways in response to nutritional and environmental stresses in yeast and mammalian cells (Hardie, 2007; Hardie and Carling, 1997; Hedbacker and Carlson, 2008). In the yeast Saccharomyces cerevisiae, the sucrose non-fermenting kinase Snf1 is a serine/threonine protein kinase that is required for derepression of the transcription of glucose-repressible genes. It is also involved in gluconeogenesis, glycogen accumulation, mitochondrial and peroxisome biogenesis, and sporulation. The mammalian ortholog of the yeast Snf1 is the adenosine monophosphate (AMP)-activated protein kinase (AMPK).
Upon cellular stress responses that deplete ATP, AMPK is activated and thus phosphorylates and inhibits the enzymes involved in cholesterol and fatty acid biosynthesis. It is well documented that AMPK can inactivate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (a key regulatory enzyme in the synthesis of cholesterol and other isoprenoid compounds), acetyl CoA carboxylase (ACC) (the rate limiting enzyme in malonyl CoA synthesis), and hormone-sensitive lipase as well. Concurrently, AMPK triggers fatty acid catabolic pathways to promote ATP production. For instance, AMPK phosphorylates and activates malonyl-CoA decarboxylase (MCD), an enzyme involved in malonyl CoA degradation. More recently, a role for AMPK in inactivating glycerol-3-phosphate acyltransferases has also been suggested in mammalian cells. AMPK, therefore, has the potential to regulate the synthesis and breakdown of triglycerides and cholesteryl esters.
Bioinformatic study of the completed sequence of Arabidopsis thaliana and rice genome revealed that, in plants, there are a large group of kinases related to the classical Snf1-type kinases from yeast. In Arabidopsis, Snf1-related kinases (SnRKs) are found on all five chromosomes and consist of 38 members. According to sequence similarity, these kinases can be classified into three subgroups: SnRK1 (three members), SnRK2 (ten members) and SnRK3 (25 members) (E. M. Hrabak et al., Plant Physiol. 2003, 132, 666-680). The structure of individual kinase is comprised of kinase domain and regulatory domain. Although these kinases show relatively high similarity in the kinase catalytic domain at the N-terminus, their regulatory domains at the C-terminus are highly divergent, which are thought to function in protein-protein interactions or regulate kinase activity (E. M. Hrabak et al., Plant Physiol. 2003, 132, 666-680). This underlines complicated functionality for each kinase.
The SnRK1 kinases, based on sequence similarity, are the closest homologues of the yeast Snf1 kinase and the mammalian AMPK. They have been isolated from a variety of species including rye, Arabidopsis, tobacco, barley, rice, sugar-beet and potato. The findings that rye and tobacco genes can complement the Snf1 mutation in yeast predict a functional similarity of plant SnRK1 genes to Snf1. Accordingly, the role for plant SnRK1 in sugar metabolism has been suggested. Antisense expression of a SnRK1 in potato resulted in the loss of sugar-inducible expression of sucrose synthase (Purcell et al., Plant Journal 1998, 14:195-202). Additional work with these anti-sense lines indicates a potential role for SnRK1 kinases for impacting carbon flow through modulating post-translation modification of ADPglucose pyrophosphorylase, a key regulatory step in starch biosynthesis (Tiessen et al., Plant Journal 2003, 35:490-500). Additional supporting evidence came from the finding that antisense expression of SnRK1 in barley resulted in little or no starch accumulation in pollen grains, causing male sterility (Y. Zheng et al., Plant Journal 2001, 28:431-441).
Unlike SnRK1 group, no representatives of the SnRK2 and SnRK3 groups are found in animals and fungi, predicting their unique regulation of cellular responses in plants. Recently, it has been shown that SnRK3 kinases, also termed as CIPKs (CBL-interacting protein kinases), can interact with a novel family of plant calcium sensors, called calcineurin B-like proteins (CBLs) (Kudla et al., 1999; Shi et al., 1999; Kim et al., 2000). In Arabidopsis, ten members exist in the CBL family, each containing three EF-hands binding to calcium (Kolukisaoglu et al., 2004). Under stress conditions, calcium signatures change and decode specific interaction between different CBL and SnRK3 (CIPK) members, leading to altered expression of the downstream genes followed by specific physiological responses. For instance, CBL1 interacts with CIPK7 and CIPK9 to promote drought response, whereas CBL9 activates CIPK3 to enhance cold response. It is noted that the CBL-SnRK3 (CIPK) network largely interacts with the plant hormone abscisic acid (ABA), which is referred to as the stress hormone because of its pivotal roles in stress responses. The evidence supporting this mechanism includes: (i) as in stress conditions, ABA can induce the expression of CBL1 and CIPK3 genes; (ii) cbl9 and cipk3 mutants are hypersensitive to ABA; and (iii) overexpression of a member of SnRK3 group (designated PKS18, corresponding to annotated At5g45820) in Arabidopsis conferred hypersensitivity to ABA during seed germination, whereas silencing of the gene resulted in ABA-insensitivity (D. Gong et al., J. Biol. Chem. 2003, 277:42088-42096).
Because of low sequence similarity in the C-terminal domains of the kinases, SnRK2 group can be further divided into two subgroups, namely SnRK2a and SnRK2b (M. Boudsocq et al., J. Biol. Chem. 2004, 279:41758-66; T. Umezawa et al., PNAS 2004, 101:17306-17311). SnRK2a consist of SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7 and SnRK2.8. The other five members, SnRK2.1, SnRK2.4, SnRK2.5, SnRK2.9 and SnRK2.10, belong to SnRK2b subgroup (Umezawa et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101:17306-11). Several studies demonstrated that individual kinases in SnRK2 subgroup may have distinct roles in biological processes. For instance, although SnRK2.2 and SnRK2.3 are two protein kinases most closely related to SnRK2.6 based on sequence similarity (Hrabak et al., 2003), they function very differently from SnRK2.6. SnRK2.2 and SnRK2.3 have been shown to be the key protein kinases mediating ABA signaling during seed germination and seedling growth. However, as a positive regulator of ABA signaling, SnRK2.6 is involved in ABA-mediated regulation of stomatal aperture, whereas seed dormancy and germination are not affected in Arabidopsis SnRK2.6 mutants (Mustilli et al., 2002; Yoshida et al., 2006).
It is believed that there exist at least three factors underscoring specialized functionality for each kinase in SnRK2 subgroup. First, individual kinases may show different temporal and spatial expression. For instance, SnRK2-8 is expressed abundantly in roots and weakly in leaves and siliques (Umezawa et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101:17306-11); SnRK2-6 is mainly expressed in guard cells and vascular tissues in Arabidopsis (Mustilli et al., 2002); and although SnRK2-2 and SnRK2-3 both display the widespread expression in various tissues, SnRK2-3 shows particularly strong expression in root tips (Fujii et al., Plant Cell, 2007, 19:485-494). Second, the regulatory domains at the C-terminus are highly divergent among different SnRK2 kinases although the kinase domains are fairly conserved. For instance, overall sequence similarity between SnRK2-4 and SnRK2-6 kinases is 70%, whereas there is only 30% identity in the C-terminal domain. This suggests that the interaction of SNRK2-6 with other signaling components may be different from that of SnRK2-4, thereby predicting different functions conferred by these two kinases. Supporting evidence came from the finding that these two kinases respond to different environmental cues (M. Boudsocq et al., J. Biol. Chem. 2004, 279, 41758-66). In addition, the role of the C-terminal domain in controlling the physiological function of the kinases was experimentally verified (Belin et al., 2006; Yoshida et al., 2006). Lastly, slight structure difference of the kinases may lead to different subcellular localization. To understand how each kinase functions, it is necessary to understand where it is localized in a living cell.